Induced Pluripotent Stem Cell Generation

Induced pluripotent stem cells (iPSCs) were generated as previously described (Holler et al., 2016 (link)). In brief, early passage fibroblast (Fig. S2C) using the following markers: Octamer-binding transcription factor 4 (Oct4; 1:400; Cell Signaling Technologies); Stage-Specific Embryonic Antigen 4 (SSEA; 1:500; Cell Signaling Technologies); Nanog homeobox (Nanog; 1:200; R&D Systems); Sex Determining Region Y-Box 2 (Sox2;1:200: ThermoScientific); Tra1–60 and Tra1–81 (1:150 each; Millipore). Correct chromosomal numbers were checked by karyotyping at WiCell (Madison, Wisconsin, USA; Fig. S2D).

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Bakkar N., Starr A., Rabichow B.E., Lorenzini I., McEachin Z.T., Kraft R., Chaung M., Macklin-Isquierdo S., Wingfield T., Carhart B., Zahler N., Chang W.H., Bassell G.J., Betourne A., Boulis N., Alworth S.V., Ichida J.K., August P.R., Zarnescu D.C., Sattler R, & Bowser R. (2020). The M1311V variant of ATP7A is associated with impaired trafficking and copper homeostasis in models of motor neuron disease. Neurobiology of disease, 149, 105228.

Publication 2020

Octamer-binding transcription factor 4 (Oct4; Stage-Specific Embryonic Antigen 4 (SSEA; Nanog homeobox (Nanog; Tra1–60 Tra1–81

Chromosomal Fibroblast Homeobox Induced pluripotent stem cells Oct4 Sox2 Stage specific embryonic antigen 4

Corresponding Organization : Barrow Neurological Institute

Other organizations : Emory University, University of Arizona

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Variable analysis

independent variables

Transduction of fibroblasts with Sendai viruses (KOS, hc-Myc, hKlf4) at an MOI of 5

dependent variables

Generation of induced pluripotent stem cell (iPSC) colonies
Expression of pluripotency markers (Oct4, SSEA, Nanog, Sox2, Tra1-60, Tra1-81)
Correct chromosomal number (checked by karyotyping)

control variables

Fibroblast passage number (
Fibroblast medium composition (10% ES-qualified FBS, 0.1 mM NEAA, 55 μM β-mercaptoethanol, high glucose DMEM)
Plating density of fibroblasts (50-80% confluency)
Coating substrates (vitronectin, Matrigel)
Culture media (Essential 8, mTeSR1)
Passaging methods (manual picking, ReLeSR)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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